The generation of the TMPRSS2:ERG recombinant bacterial artificial chromosome (BAC) using recombineering has been completed. Recombineering relies on the process of recombination between homologous regions, one end of which is a linear DNA fragment. We have made novel use of this technique to precisely combine two human genomic BAC clones encoding 25 Kb of the TMPRSS2 promoter and its first 2 exons with 167 Kb of ERG containing exons 4-11 and the entire 3UTR. In this way, we have replicated this common genomic translocation and maintained important regulatory information in both the promoter/enhancer of TMPRSS2 and in the intronic and 3UTR regions of ERG, a gene that is subject to a variety of alternative splices. We believe that maintaining the regulatory properties within the translocated DNA segments is crucial for analyzing expression characteristics and functional consequences with respect to various developmental lineages within the prostate. The production of TMPRSS2:ERG construct required slightly longer than 12 months because the unique use of recombineering for our purposes introduced several steps that necessitated trouble-shooting. The design of the 230 Kb construct was crucial and demanded extensive bioinformatics analysis of the parent BAC clones. In addition, the homology arms needed to be optimized for efficient recombination. Several cloning and two recombineering steps were involved in generating a construct consisting of the TMPRSS2:ERG fusion. (A) A base vector was constructed which contains a neomycin cassette and two pairs of homology arms necessary for recombineering. (B) The first recombineering event involved removal of the TMPRSS2 promoter/enhancer region and the first two exons from its BAC and placement in a pBR322 vector. (C) The second recombineering event involved placement of the TMPRSS2 region in front of the ERG gene in its own BAC. As this step required electroporation and recombination of a 25 Kb linear fragment, special transfer, selection, and screening conditions were developed. The correct structure of the recombinant BAC was verified using restriction digestion and pulse field electrophoresis as well as PCR mapping. The Laboratory of Animal Sciences Program at NCI Frederick has generated transgenic mice using the TMPRSS2:ERG BAC. In total 9 FVB founders and 6 C56/BL6 founders have exhibited germ line transmission. Analysis of these strains for expression of the human ERG transgene has established expression in several of the lines. Western blot analyses have established that a protein of the expected size is produced.